title: Next-generation sequencing of the Chinese hamster ovary microRNA transcriptome: Identification, annotation and profiling of microRNAs as targets for cellular engineering.
abstract: Chinese hamster ovary (CHO) cells are the predominant cell factory for the production of recombinant therapeutic proteins. Nevertheless, the lack in publicly available sequence information is severely limiting advances in CHO cell biology, including the exploration of microRNAs (miRNA) as tools for CHO cell characterization and engineering. In an effort to identify and annotate both conserved and novel CHO miRNAs in the absence of a Chinese hamster genome, we deep-sequenced small RNA fractions of 6 biotechnologically relevant cell lines and mapped the resulting reads to an artificial reference sequence consisting of all known miRNA hairpins. Read alignment patterns and read count ratios of 5' and 3' mature miRNAs were obtained and used for an independent classification into miR/miR* and 5p/3p miRNA pairs and discrimination of miRNAs from other non-coding RNAs, resulting in the annotation of 387 mature CHO miRNAs. The quantitative content of next-generation sequencing data was analyzed and confirmed using qPCR, to find that miRNAs are markers of cell status. Finally, cDNA sequencing of 26 validated targets of miR-17-92 suggests conserved functions for miRNAs in CHO cells, which together with the now publicly available sequence information sets the stage for developing novel RNAi tools for CHO cell engineering.
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 19, A-1190 Vienna, Austria.
authors.forename: Matthias
authors.initials: M
authors.lastname: Hackl
authors.forename: Tobias
authors.initials: T
authors.lastname: Jakobi
authors.forename: Jochen
authors.initials: J
authors.lastname: Blom
authors.forename: Daniel
authors.initials: D
authors.lastname: Doppmeier
authors.forename: Karina
authors.initials: K
authors.lastname: Brinkrolf
authors.forename: Rafael
authors.initials: R
authors.lastname: Szczepanowski
authors.forename: Stephan H
authors.initials: SH
authors.lastname: Bernhart
authors.forename: Christian
authors.initials: C
authors.lastname: Höner Zu Siederdissen
authors.forename: Juan A Hernandez
authors.initials: JA
authors.lastname: Bort
authors.forename: Matthias
authors.initials: M
authors.lastname: Wieser
authors.forename: Renate
authors.initials: R
authors.lastname: Kunert
authors.forename: Simon
authors.initials: S
authors.lastname: Jeffs
authors.forename: Ivo L
authors.initials: IL
authors.lastname: Hofacker
authors.forename: Alexander
authors.initials: A
authors.lastname: Goesmann
authors.forename: Alfred
authors.initials: A
authors.lastname: Pühler
authors.forename: Nicole
authors.initials: N
authors.lastname: Borth
authors.forename: Johannes
authors.initials: J
authors.lastname: Grillari
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D017124:Conserved Sequence / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D005818:Genetic Engineering; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D022661:RNA, Untranslated / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods*; D014158:Transcription, Genetic
chemical_list: D035683:MicroRNAs; D022661:RNA, Untranslated